The process of X-inactivation in mammals occurs by the formation of facultative heterochromatin, a phenomenon central to normal development and abrogated in some cancers. In this process, an accumulation of stable XIST RNA structurally associates with one X chromosome in females and initiates a cascade of chromosome remodeling that silences the inactive X (Xi), forming a heterochromatic Barr Body. We now need to know how XIST RNA localizes to and "paints" its parent chromosome, and how this leads to the structural transformation and condensation of a whole chromosome. Our approach to these questions utilizes molecular, biochemical and structural analyses, coupled with bioinformatics of genomic sequence organization. Our aims deal with distinct but inter-related aspects of the functional and structural transformation of the chromosome, focusing on the interaction of XIST RNA with the chromosome and the potential role genomic repeat sequences. Aim 1 builds on our recent success in manipulating XIST RNA localization, to better understand the regulation of XIST binding, specific factors involved, and extend strong preliminary results on specific [unreadable] histone modifications, as well as heterochromatin factors and scaffold attachment factors implicated in Xi. The factors involve may provide insight into broad heterochromatic instability in cancer, of which Xi/XIST defects may be one hallmark. Aim 2 investigates a novel model for silencing of an entire chromosome, where XIST RNA is not acting at a local or individual gene level, but has a more architectural relationship with the whole interphase chromosome territory, particularly with an inner core enriched in repeat elements. In this model, XIST first interacts with the repeat-rich regions of the X chromosome, nucleating a heterochromatic core that then propagates to the more peripheral protein coding genes. In Aim 3, the relationship of sequence context to escape from silencing is examined in a region that consistently escapes silencing, using a systematic transgene approach, combined with bioinformatics and molecular cytological analyses. Our studies will focus largely on human X inactivation, which has been less well studied, using somatic cell, transgene and human ES cell models. Understanding what establishes and maintains human Xi heterochromatin has relevance to [unreadable] heterochromatic instability in cancer as well as formation of facultative heterochromatin in embryonic cells, and thus our studies have significant clinical implications. [unreadable] [unreadable] [unreadable]